Process for cultivating aerobic microorganisms



United States Patent 2 Claims. (or. 195-109 The present invention relates to an improvement of a process for the pure cultivating of large amounts of aerobic microorganisms. More particularly, the present invention relates to a process for preparing large amount of the pure culture of aerobic microorganisms while feeding a large amount of sterile air for a long period of time, said sterile air being prepared by use of a special means for removing microorganisms.

It is well known to produce food, medical articles, articles for the industrial use and others by cultivating microorganisms. According to present practice aerobic bacteria are cultivated by means of forced aeration and fermentation-production are efficiently and economically effected. However, one of the weak points of the abovementioned process is that there is always a danger of making all of the product waste when the yield is lowered by infection of infectious microbes. Therefore, microorganisms or microbes employed for the production shall be prevented from mixing with infectious microbes, and the purest culture shall be carried out. Furthermore, the inside of a container employed for the production and the medium employed shall be previously sterilized. Particularly, in case of aerobic bacteria, much amount of air to be fed is required. However, a large amount of infectious microbes and spores float in the air as shown in the following Table 1, so that great labour is required for making air sterile air.

Table l.TEST FOR COUNTING NUMBERS OF INFECTIOUS MICROBES IN THE AIR Numbers of infectious Place Time microbes Reporter (per cubic meter of air) Tokyo The early years of Report by Eisei the Era of Meiji. Shiken-jo. A city of 19 39 10 I. Eng. Ch.

England. (1956). In general 2X10 Mr. Hamiree.

(1) Processes for sterilization 1-1. Chemical processes (oxone, bactericidal gases and others) 1-2. Physical processes (heating, ultrasonic wave and others) ( 2) Process for removal of bacteria:

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2-1. Processes of filtration (carbon powder, cotton, glass wool and others) 2-2. Processes of adsorption (electric method, washing with water and others) Among these processes, the most general processes are 2-1, and processes of 22 may be employed together, from the standpoints of cost of the apparatus and practical conditions, such as difficulty of treating. A process of 2-2 is seldom used, singly.

Processes of 21 are characterized in that filter mediums are used. As filter mediums there are various kinds of materials. One of the most effective filter mediums is glass wool, which has the highest activity of removing infectious microbes and is rich in durability. However, even in this case, the amount of the filter medium to be used is considerable and a large space area is required. Moreover, the usable period of glass wool is at most one year, when it is continuously used, and much labour is necessary for exchanging it with new one.

The present inventors have compared many materials with glass wool, and as the result found that the pure cultivating of large amount of aerobic microorganisms may be easily and certainly carried out by feeding air from which infectious microbes are removed by passing air through layers of sponges consisting of acetal compounds mainly composed of polyvinyl alcohol and having holes of 2200/L in diameter, said layers of sponges being manufactured in such a way as they have resistance against heat of about C. to C.

It has been known that sponges consisting of acetal compounds of polyvinyl alcohol are used for removal of dust. However, removal of dust is different from removal of bacteria in the following points.

(1) Most parts of dust to be removed are fine dust of 10 1. in the diameter. On the other hand, infectious microbes and sporges of about Lu. in the diameter become the object.

(2) Complete removal of dust in the air is not necessary, but in case of aeration for the culture, removal in an amount of 100 percent is ideal. Because, if merely a few infectious microbes are mixed into the culture medium, fission is immediately begun and they propagate to great numbers in a short period of time.

Therefore, it is very dangerous to use filter mediums employed for preventing dust in general for removal of microorganisms. Whether said filter mediums can be used or not, and how to use them can not be decided until exact bacteriological and fermentation engineering studies are made.

(3) A filter medium to be used for removing bacteria in the culture under aeration itself, shall be sterilized in many cases. However, in case of a filter medium to be used for the purpose of preventing dust it is unnecessary. In the case where a culture of aerobic microorganisms is eifected under aeration, the culture medium, the container and the inner walls of pipes connecting to said container shall be sterilized every time whenever air is fed. Moreover, the inside of an air filter is required to be sterilized, too. The sterilization is carried out by means of steam at a high temperature, so that the filter medium should have durability against steam of a high temperature.

In general, sponges mainly composed of polyvinyl alcohol, a kind of filter mediums for removing dust which are in the market, are immediately softened by hot steam, and lose the form and the function of filter mediums. Therefore, they can not be used. If sponges, mainly composed of polyvinyl alcohol, are processed in such a way as they have resistance against heat of from about 100 C. to 130 C. by means of acetalation with dialdehyde and the like, heat treatment or treating with suitable resin or both of those treatments, the weak point is gotten rid of and the object will be attained. Further, it is known that polyvinyl alcohol forms spongelike materials under conditions in the case of acetalation hol do not change so much and have unchanged capacity of removing. infectious microbes even though they are used for 2 years. So that, it can be judgedthat they may be used for a further longer period.

of its aqueous solution, and forms porous materials ca- 5 From these experiments it may be clarified that sponges pable of passing liquid materials well according to conmainly composed of polyvinyl alcohol is remarkably suditions such as addition agents and others. (Refer to perior in, also, durabilit to glasswool.

Japanese Patents Nos. 184,298, 196,838, and 196,415.) In these experiments it has'been confirmedthat sponge Sponges in form of a plate having holes of 2200a layers, mainly composed of polyvinyl alcohol are ,desirin the diameter, which have been producedv by giving able to satisfy the following conditions thermal resistance lest they should be softened at a tem- (1) They have thermal resistance against heat (in par-' perature'ranging from about 100 C. to 130 C., may ticular heating with steam) ran'gingfrom 100 C. to. remove bacteria remarkably efiiciently, compared with 130 C. glass wool conventionally employed. It will be more (2) The diameter of a hole .is'suitable to be 2-200a concretely explained by the following experimental exor so. In case of a hole having the diameter of less amples. In this case, windpressure which is given at than 2a, aerationis badly carried out, and in case of a. the time of aeration can be well dealt with by use of a hole having the diameter of more than 200a no effect suitable auxiliary apparatus. Therefore, pressure-resistcan be attained. ance of sponges is not of such a degree as being partic- If the layer is rough, itis' necessary to increase the ularly noticed. thickness 21 little or to use several layers at one time.

y l (3) As for thickness of a layer'employed, it may be:

Experimental Example ].C0mparzson 0f P J f more than about 0 of the thickness of glass wool, in removing bacteria of glass Wool wlth thai of sponges the case where it is used at' the same wind velocity. mainly composed of polyvinyl alcohol (4) Against wind pressure, a suitablereinforcing ap- Compressed i (15 k j y) i i d d from a 25 paratus inform of a wire-netting or'others is desirable compressor into a filter tube of 20 cm. in the diameter to be employedfilled with glass wool or sponge plates, mainly composed 111 case of Putting on the filter medium; it d6? of polyvinyl alcohol. Then, compressed air' is passed sirable to tighten by a suitable means, for exampleI :-by through said glass wool or sponge plates and the effect pa in Order to Prel/(mt leakage from the ysllfromlde of removing bacteria is examined in each case. The reingssults are shown. in Table 2. However linear velocity EXAMPLE 1 0f thefiefatlon 1S 10 CIYL/sec- The p y 6f removing A fermenter made of stainless steel, having the capac-' bacteria is examined from the degree of generation ofniity f 600 liters, and equipped with a sparger foraera-v fectious microbes after aeration is effected for 7 days W tion and a stirrer in the inside, and a compressor, a pipe", in the sterilized culture medium. and a filter. tube of 500 mm. in the outside diameter and TABLE 2 Thickness of Lowering Kinds of filter the layer pressure Capacity of removing medium Filter mediums 0t filter of air, infectious microbes medium, percent Glass wool (eddy A yarn having 10 in the 3 Generation of infectious miform). diameter. crobes.

D0 do 100 6 No generation of infectious microbes. Sponges, mainly com- A hole having 10 in the 0.3 0 Do.

posed of polyvinyl diameter. alcohol.

Do A hole having 60p. in the 2 0 D0.

diameter.

The tests are repeated 10 times, but the same results 500 mm. of height is used. Compressed air is introduced are attained after all. From the results, it can be underthrough said pipe and filter tube from the compressor to stood that sponges, mainly composed of polyvinyl alcohol the fermenter. A sponge layer having holes of 101:; in having merely 0.3 cm. in thickness have bacteria-removing di et d 1 i thi k e d 5 ponge l f activity corresponding to that of glass W001 100 having holes of in diameter and.5 mm. in thickness in thickness and moreover do not lower pressure of air. are put on in the inside of the filter tube, Said sponge. Experimental Example 2.C0mparis0n of durability of layers Processed such a Way as havingheatp s glass wool with that of sponges mainly composed of ance, Whlch are 111311113 Composed 'p y alcohol? polyvinyl l h l The outlet of the filter tube is connected with the sparger I th t e 1 ed EX r.menta1 60 in the fermenter through a pipe.

n e Same appara us as mp 0y 1 Fe l 350 liters of fermentation liquor is fed in the'fermenter Example 1, each of glass wool and said sponges lS steriv and steam 1S blown in. The temperatures of the inside lized by use of steam for 1 hour every time whenever f h filt t b d th t a f V th filt the apparatus is used. The operation is repeated four 9 t 6 er u e an e pipe 1 8 mm 6 times in a month. The physical properties of the filter {Elbe to the Sparger the fermentauon'liquor andvihe 3' mediums are compared with each Othen Thermal s de of the fermenter are raised to 120 C. Aftersteri sistance of sponges employed in the above case have ilzatlon by heatmg for 1 hour 1t 15 stopped w been previously strengthened by means of acetalation Then thg temper'flture q l 1; or heat treatment. The diameter of a hole of the sponges lowered to 300 Whlle feedmg bactena F T1 ..P is 10,u.. Heating with steam is effected at 120 C. through the filter tube and an elevated pressure. Mold.

After 12 months, glass fibers are aerated and chan starters of Aspergillus niger, which have previously been i i Further, h become weak and a part f prepared by pure culture,areinoculate ,d under thegbacteria the becomes poor de y Th f h can t b d free condition. Thereafter, cultivation iscarried out withfor the purpose of removing infectious microbes. On the stirring and aeration. After aboutg72 hours, good liquid other hand, sponges mainlycomposed of polyvinyl alco- 75 koji (submerged mold amylase) is obtained. In, that case, no infectious microbes other than live mycelia of Aspergillus niger can be seen in the culture medium.

Furthermore, the present inventors have found that polyvinyl alcohol acetal sponges which are given resistance against hot steam by processing with synthetic resins have quite efiicient activity for removing bacteria, compared with conventional filter mediums for removing bacteria. That is, the present invention relates also to a The results are shown in the following table. That is, a layer of polyvinyl acetal sponge processed has the activity of the same degree even though its thickness is 0.3-2% of the thickness of a glass fiber. No reduction of pressure of air can be seen in case of a sponge layer.

Furthermore, the sponge layer has resistance against hot steam, and therefore good liquid koji (submerged mold amylase) can be fermented and produced.

Thickness Lowering Generation Kind of filter medium Filter medium of layers of pressure of infectious of filter of air microbes medium (percent) Glass fiber A fiber has a diameter of 10p 50 3 Do "(10 100 6 Sponge processed. with melamine A hole has a diameter of l0;r 0.3 0

resin. Sponge processed with phenol A hole has a diameter of 60 2 0 resin. Sponge processed with furan A hole has a diameter of p 1 0 resin.

We claim:

process for the culture of aerobic microorganisms, characterized in feeding sterile air prepared by treating air with a polyvinyl acetal porous material which is given resistance against hot steam by processing with synthetic resins.

The manufactured sponge used in the present invention, of which thickness as a filter layer is less than 1% of that in case of glass fiber, has the same or stronger activity of removing bacteria in comparison with that of glass fiber. The filter medium is cheap, and the required space area is small. Aeration resistance can hardly be seen. Moreover, the period of using said sponges continuously in sterilization with steam is the same with that of glass fiber, and it is easy to exchange this filter medium.

As synthetic resins for processing sponges, for example, melamine resin, phenol resin, furan resin and the like are especially excellent. In order to carry out particularly advantageously the process for giving sponges resistance against hot steam, the porous materials are dipped in a 550% solution of synthetic resins, and then the solution is removed. After the treated porous materials are dried, the materials are treated at a high temperature ranging from 100 C. to 200 C. Other means may be employed.

EXAMPLE 2 In 5 fermenters having the capacity of 600 liters equipped with a sparger for aeration and a stirrer, and connected with a pipe for introducing compressed air. On the way of the pipe, a filter tube having 500 mm. in the inside diameter is provided. Into filter tubes glass fibers in form of eddies and manufactured polyvinyl acetal sponges are placed in such a way as air fed to the fermenters passes through these filter mediums.

Into each of five fermenters, 350 liters of fermentation liquor is fed. Then, steam is blown into the insides of the filter tube and the fermenter. The fermentation liquor and the insides of the fermenter and the filter tube are heated to 120 C. and sterilized for minutes. Then, it is stopped to blow steam in. They are cooled with feeding compressed air. Mold starters of Aspergillus niger are inoculated under the condition free from bacteria. Thereafter, culture is carried out at 28 C. for 72 hours, while stirring and aerating.

1. In a process for cultivating aerobic microorganisms by fermentation in industrial scale employing purified and sterilized air, the improvement comprising purifying the air by passing the air through a spongy material consisting essentially of a polyvinyl acetal having holes of sizes ranging from about 2 to about 200 microns which has been treated with a thermosetting artificial resin selected from the group consisting of melamine resin, phenol resin and furan-resin whereby resistance to steam at temperatures ranging from about C. to C. is imparted to said spongy material.

2. In a process according to claim 1, wherein said spongy material is prepared by dipping spongy polyvinyl acetal in a 5-50 percent solution of a thermosetting resin selected from the group consisting of melamine resin, phenol resin and furan resin, removing said solution, drying the treated resin, and subjecting said treated resin to heat at a temperature ranging from about 100 C. to 200 C.

References Cited by the Examiner UNITED STATES PATENTS 2,298,561 10/ 1942 Hendrickson 109 2,556,295 6/1951 Pace 55528 2,627,494 3/ 1953 Keko et al. 19596 2,654,440 10/ 1953 Robinson 55-524 2,689,199 9/1954 Pesce 55528 2,766,176 10/1956 Jeffreys 19596 2,827,395 3/1958 Jordan et al. 17798 2,882,997 8/1959 Smith et a1 55524 2,920,718 1/1960 Howell et a1 55524 3,013,950 12/1961 Gavin 195142 3,075,888 1/1963 Achorn et al. 195-139 3,096,204 7/1963 Spangler et al 11798 OTHER REFERENCES Rose: The Condensed Chemical Dictionary, Reinhold Pub. Corp., New York, 5th ed., pages 884 and 885, 1956.

A. LOUIS MONACELL, Primary Examiner.

A. E. TANENHOLTZ, Assistant Examiner. 

1. IN A PROCESS FOR CULTIVATING AEROBIC MICROORGANISMS BY FERMENTATION IN INDUSTRIAL SCALE EMPLOYING PURIFIED AND STERILIZED AIR, THE IMPROVEMENT COMPRISING PURIFYING THE AIR BY PASSING THE AIR THROUGH A SPONGY MATERIAL CONSISTING ESSENTIALLY OF A POLYVINYL ACETAL HAVING HOLES OF SIZES RANGING FROM ABOUT 2 TO ABOUT 200 MICRONS WHICH HAS BEEN TREATED WITH A THERMOSETTING ARTIFICIAL RESIN SELECTED FROM THE GROUP CONSISTING OF MELAMINE RESIN, PHENOL RESIN AND FURAN-RESIN WHEREBY RESISTANCE TO STEAM AT TEMPERATURES RANGING FROM ABOUT 100*C. TO 130*C. IS IMPARTED TO SAID SPONGY MATERIAL. 